Electrophoresis of large charged molecules, such as nucleic acids, deoxyribonucleic acid (DNA) fragments, or proteins has been an established laboratory method for approximately 25 years. In its most common form, an electric field is imposed between opposite ends of a thin slab of gel. A sample, containing a mixture of various charged molecules is introduced into one end of the gel. The electric field causes the molecules to migrate to the opposite end of the gel. The velocity at which each substance of interest migrates is dependent upon its mobility, a characteristic determined by the length of the substance, shape, and other characteristics. Molecules having similar mobility will migrate as a band at a given velocity. With time, the sample will be separated into distinct bands, each composed of like molecules.
By means of staining or radioactive tagging, the various nucleic acids or proteins of interest can be located and identified. As described above, electrophoresis is a simple and sensitive analytical technique. The recovery of the nucleic acids or proteins from the gel following electrophoresis has historically been difficult. Several methods have been tried for recovering these substances.
For example, one method is the crush and soak method which involves excising the gel material containing the substance to be recovered from the gel slab and grinding or crushing the gel material. The crushed gel material then is soaked in a salt solution for long periods of time. This extracts the desired protein or nucleic acid from the gel. The substance then is ethanol precipitated from the salt solution. This process is quite slow and is not efficient for most purposes.
Another method for recovering substances from gel material known in the art is the melting agarose/chemical extraction method whereby the gel material containing the nucleic acid or protein to be recovered is excised from the gel slab and is melted at about 65.degree. C. Phenol extraction is followed by organic chemical purification. The recovery efficiency by this method is relatively low and the purification procedures are long and labor intensive.
The blitz blotting method of recovery entails placing the entire gel slab in contact against a sheet of DEAE cellulose paper, which is known for its ability to bind DNA fragments. This sandwich then is immersed in a buffer solution and by electrophoretic means, all the bands are transferred from the gel to the DEAE paper. This method has a relatively high recovery efficiency, but the apparatus is costly and cumbersome. Another method using DEAE involves cutting slots in the gel immediately downstream of each of the bands. DEAE paper is inserted into the slots and by means of electrophoresis, the bands are driven into the DEAE paper. This method is extremely technique dependent. Cutting the slot deforms the electric field which causes varying portions of the band to be driven around the slot.
The gel decomposition/glass beads method of recovery requires decomposing the gel material containing the material of interest by chemical means. The resulting mixture, in a buffer, is poured over glass beads which are removed and washed. The nucleic acid or protein is eluted from the glass beads with a salt solution. The method results in fair recovery efficiency with large molecules, but buffer conditions are very critical.
U.S. Pat. No. 4,552,640, issued to Kartenbeck (1985), describes an electrophoretic apparatus for the quantitative elution of proteins or polypeptides from a gel which includes an upper chamber for holding a buffer solution containing the gel from which the proteins or polypeptides are to be eluted. An upper electrode is provided in the upper chamber. A lower chamber for holding a buffer solution is disposed beneath the upper chamber and includes a lower electrode. A septum separates the upper chamber from the lower chamber. A connecting passage in the septum connects the upper and lower chambers. A collecting capsule for the proteins or polypeptides is formed from a dialysis membrane material and is disposed at the end of the connecting passage in the lower chamber. The capsule is adapted to be suspended in the buffer solution which is to be held in the lower chamber.
The Kartenbeck apparatus suffers from several disadvantages. The dialysis membrane, due to its relatively large surface area, can adversely adsorb the very material sought to be recovered. Also, the relatively large volume of the lower chamber can cause undue dilution of the sample.
U.S. Pat. No. 4,545,888, issued to Walsh (1985), describes an apparatus for the recovery of nucleic acids and other substances which consists of a plurality of transfer chambers suitably supported in a vessel for containing an aqueous buffer solution, a plurality of filter discs for support of a layer of DEAE cellulose resin in the bottom of the transfer chambers, a plurality of negative electrodes, a positive electrode for placement in the buffer which will surround the plurality of transfer chambers, and a power supply. A gel slab containing nucleic acids is placed into the transfer chambers. When an electric current is passed through the chambers, the nucleic acids migrate to the positive electrode. On passing through the DEAE, the nucleic acids are brought into intimate contact with the DEAE resin and are bound to the surface of the DEAE resin. The nucleic acids are recovered by established elution procedures. This process requires an extra elution step and thus, is not efficient.
U.S. Pat. No. 4,699,706, issued to Burd (1987), also describes an electrophoretic method and apparatus for extraction of species from electrophoresis media. This method involves placing a separation medium in a tube closed at one end by a retaining material such as a glass frit, and sealing the closed end of the tube against the mouth of a receiving cup which has a semipermeable membrane for its bottom. The tube with receiving cup attached is inserted in a tube electrophoresis cell such as those commonly used in analytical laboratories. As current passes through the tube, the molecular species migrates from the separation medium to pass through the retaining frit and collect on the membrane for subsequent recovery. The structure used for recovery in this method is complex since the dialysis membrane must be held in place by a retaining ring, a gasket and internal shoulders. No provision is made for capping the column to hydraulically isolate the sample collected in the sample cup. Removal of the sample cup may lead to the disruption of the collected sample because of leakage through the frit or due to excess fluid in the sleeve holding the cup. Further, the recovery is inefficient since the dialysis membrane is smaller than the diameter of the frit.
There is a need in the art for a simple, convenient, inexpensive apparatus and method for elution of proteins, enzymes or nucleic acids from gels used in electrophoresis.